Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-beta1 and TGF-beta receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. PAPERCLIP:

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model. Induced nuclear reprogramming through induced pluripotent stem cell (iPS)2 technology is an amazing achievement full of challenge to the intellect and important practical implications (1, 2). Overexpression of exogenous factors that are highly enriched in embryonic stem cell (ESC) can rearrange the genetic program of different cell types, including somatic and adult stem cells, and induce a long lasting ESC-like pluripotent state (3-7). The repercussions of iPS technology are vast: it provides a way to create patient-specific stem cells that bypasses ethical and technical issues surrounding human ESC derivation and somatic cell nuclear transfer (8, 9), a state of the art model for studying genetic diseases in vitro (10, 11), and an incredible backwards route that can crystallize our current understanding of developmental and stem cell biology. Many questions, especially mechanistic, remain unanswered, but the current rhythm of research may bring iPS to clinical application sooner than expected. However, before jumping onto such extraordinary endeavor, safety must be scrupulously tested in an animal model close enough to humans. Nowadays that iPS technology is expanding, with improved delivery systems, chemical additions, new tissue culture conditions, and multiple cell sources being reported regularly, such animal model is essential to set up quality standards (12-18). Mice, and maybe rats, will possibly continue unrivalled as the easier ways to learn about reprogramming machinery and improve methodology, but their size, physiology, and reduced lifespan are handicaps for making serious assumptions regarding safety in humans. Given philogenetic similarity, monkeys are theoretically an excellent alternative, but in practice ethical concerns remain to at least some extent, and they are neither easy to maintain nor to breed. Swine, a regular source of food whose farming humans have adapted over myriads of years and whose physiology is r...

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation 1. EPSCs had enriched molecular signatures of blastomeres and possessed the developmental potency for all embryonic and extraembryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras, and produce primordial germ celllike cells in vitro. Under similar conditions, human ESCs and iPSCs can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Significantly, trophoblast stem cell-like cells can be generated from both human and porcine EPSCs. Our pathwayinhibition paradigm thus opens a new avenue for generating mammalian pluripotent stem cells, and EPSCs present an unique cellular platform for translational research in biotechnology and regenerative medicine.